Production of amino acids

ABSTRACT

AEROBIC FERMENTATION PROCESS FOR THE PRODUCTION OF EXTRACEULLULAR AMINO ACIDS WHICH USES, AS A PRIMARY SOURCE OF CARBON, A C1-C30 N-ALIPHATIC HYDROCARBON FEED WITH AN AQUEOUS, INORGANIC SALT GROWTH MEDIUM, AND A MICROORGANISM CAPABLE OF GROWTH ON SAID N-ALIPHATIC HYDROCARBON FEED, SAID PROCESS BEING CONDUCTED IN THE PRESENCE OF CELLULOSE. IT IS PREFERRED THAT THE N-ALIPHATIC HYDROCARBON BE THE SOLE SOURCE OF CARBON IN THE PROCESS, PREFERRED N-ALIPHATIC HYDROCARBON FEED BEING COMPRISED PRIMARILY OF C11-C30 N-PARAFFINS.

Feb. 9, 1971 80ml 0 Saline Solution 2.0ml of Substrate A" J. D. DOUROS, JR ETAL 3,562,110

PRODUCTION OF AMINO ACIDS Filed Jan. 25, 1968 STOPPER l GLASSTUBING 3 CELLULOSE BAG 4 20 ml of a 5X ComLP 5 Swims Medium J. D. DOUROSQ Jr. L. A. NASLUND invert-em W. J. LAHL :7 W, By W001 {I I Pg? AN army United States Patent O 3,562,110 PRODUCTION OF AMINO ACIDS John D. Douros, Jr., Littleton, Colo., Lars A. Naslund, Morganville, N.J., and William J. Lahl, Marysville,

3,562,110 Patented Feb. 9, 1971 ice amino acids obtained have been low and the rate of accumulation of the product very slow.

Another recent, and even more promising, technique for biologically synthesizing extracellular amino acids and Ohio, assignors to Esso Research and Engineering Comfood Protelll 18 by cultivating the microorganisms on pe- Pany, a corporation of Delaware troleum substrates. This type of fermentation 1s usually Filed Jan. 25, 1968, Ser. No. 700,580 conducted 1n an aqueous b1osynthes1s bath containing a Int. Cl. C12d 13/06 hydrocarbon feed, an inoculant of the microorganism to US. Cl. 19528 5 Claims be grown, an aqueous growth medium, oxygen, nitrogen and other indispensable nutrients. This technique allows the use of hydrocarbon feeds, which are widely available ABSTRACT OF THE DISCLOSURE in the necessary quantities and are less expensive than car- Aembic f tatig Process f h production of bohydrates. It is also knOWn to use various biological cata extracellular amino acids which uses, as a primary source lysts 111 fel'menfatloll'proflessesbiosynthetic Process f carbon, a c c li h i h d b f ed i h of the present mvention is applicable to the biosynthesis an aqueous, inorganic salt growth medium, and a micro- Of all m1CfP0fgI11SmS Whlch are Capable of growth on organism capable of growth on said n-aliphatic hydro- 1- 3!) p f hydrocarbon feeds,part1cu1ar1y lr so carbon feed, said process being conducted in the presence 'p f del'lved m p p f hydrpcarbon fractlonsof cellulose. It is preferred that the n-aliphatic hydrocar- While the Present lnveljltlon pp i to a broad bon be the sole source of carbon in the process, preferred scope of operfflble bactfllal ml'cfoofganlsms, there are li h h d b f d b i comprised i il a number of microorganisms WhlCll are especially suitable f CircaJ ffi for hydrocarbon assimilation, These microorganisms are tabulated hereinbelow, along with their corresponding ATCC registration numbers which were secured by depositing samples with the American Type Culture Collec- Thus, the present invention is broadly concerned with tion, 212 M Street, Northwest, Washington 7, DC, or a biosynthesis fermentation process for the production of other designated numbers.

TABLE I 1 Division Class Order Family Tribe Genus Species No. ATCC 2 Protophyta-Schizomycetes Pseudomonada1es-Pseudomonadaceae Pseudom onas Ligustri 15522 3 Pseudomallei- 15523 4 Orville 15524 5 -Eubacteria1es -Micrococcaceae Micr:ococcus Cerificans -14987 6 L-Sarcinar-Sp QHBlSlB Natick 7 Brevibacteriaceae Brexiibacterium-Insectiphilium15528 a -Hea1i1'-l5527 9 Achromobac Alca Sp 15525 10 orynebactetiaceae- -Ce11umonas- ---Ga1ba 15526 11 orynebacterium Sp 15529 12 Pausomet-abo1um15530 13 Arthtobacter- Simplex 6946 15 -Actinomycetales Mycobacceriaceae MycobacteriumRhodchx.-ous

16 -Actinomycetaceae locar'dia Sp 1? LSP high concentrations of extracellular amino acids, as well as using the residual cells for animal and human foods. More particularly, the invention is concerned with the use of C -C n-aliphatic hydrocarbon feed, particularly C -C n-parafiins as the primary source of carbon in the process. In accordance with this present invention, C C n-paraffins are contacted with a microorganism under fermentation conditions in the presence of cellulose to produce high yields of extracellular amino acids. This process may be carried out continuously or batchwise.

Heretofore, the microbiological production of amino acids, such as lysine, glutamic, isoleucine, valine, and the like, has reqiured the use of expensive carbohydrate substrates and precursors which had to be added to the fer mentation broth. Moreover, the yields of extracellular The Micrococcus cerificans (14987), which was isolated and identified by Dr. R. E. Kallio et al., Journal of Bacteriology, vol. 78, No, 3, pages 441-448 (September 1959), is particularly desirable. Further identification is as follows:

MORPHOLOGY 3 GRAM REACTION Negative.

Colonies on defined agar are small (1 mm.), circular convex, having entire edge. Colonies on nutrient agar are larger (2 to mm.), raised mucoid, generally round.

Within a species there can be many different strains comprising variations and both natural and induced mutants.

The morphology and growth reaction characteristics of other organisms listed above are given in US. Pat. 3,308,035 issued Mar. 7, 1967 entitled, Process for Producing a High Protein Composition by Cultivating Microorganisms on an N-Aliphatic Hydrocarbon Feed, inventor John D. Douros, Jr.

The growth media comprise an aqueous mineral salt medium and excess oxygen. Oxygen is supplied to the cultivation substrate medium or broth in any form capable of being assimilated readily by the inoculant microorganism. Oxygen-containing compounds may also be used to supply oxygen as long as they do not adversely affect microorganism cell growth and the conversion of the oxidized hydrocarbon feed to microorganism cells. Oxygen may be supplied as an oxygen-containing gas, such as air at atmospheric or elevated pressure or oxygenenriched air wherein the oxygen concentration may be up to 70% to 90%. In general, between about 0.1 and about 10, preferably between about 0.8 and about 2.5 volumes per minute of air are supplied to the reactor per volume of liquid in the fermentor.

Nitrogen is essential to biological growth. The source of nitrogen may be any organic or inorganic nitrogencontaining compound which is capable of releasing nitrogen in a form suitable for metabolic utilization by the growing microorganism. Suitable organic nitrogen compounds are, for example, proteins, acid-hydrolyzed proteins, enzyme-digested proteins, amino acid, yeast extract, asparagine, and urea. Suitable inorganic nitrogen compounds are ammonia, ammonium hydroxide, nitric acid or salts thereof, such as ammonium phosphate, ammonium citrate, ammonium sulfate, ammonium nitrate and ammonium acid pyrophosphate. A very convenient and satisfactory method of supplying nitrogen to the process is to employ ammonium hydroxide, ammonium phosphate or ammonium acid phosphate, which can be added as the salt per se or which can be produced in situ in the aqueous fermentation media by bubbling ammonia gas or gaseous ammonia through the broth or injecting aqueous ammonium hydroxide into the broth to which phosphoric acid was previously added, thereby forming ammonium acid phosphate.

In this way the desired pH range of about 3.0 to 8.5 is maintained and the requisite nitrogen supplied. If the microorganism comprises a yeast the preferred pH is in the range of 3.0 to 7.5 such as 4.0 to 5.0. If the microorganism comprises a bacteria the desired pH is in the range of 5.0 to 8.5, such as about 7.0. Ammonium hydroxide may be supplied to the biosynthesis bath in amounts of between about 0.08 and about 0.20, preferably between about 0.1 and about 0.15, gram of nitrogen per gram of dried cells produced. This amounts to between about 0.01 and about 1.0 wt. percent, preferably between about 0.1 and about 0.15 wt. percent, nitrogen based on the total biosynthesis bath.

In addition to the oxygen and nitrogen, it is necessary to supply requisite amounts of selected mineral nutrients in the feed medium in order to insure proper microorganism growth and maximize the assimilation of the oxidized hydrocarbon by the microorganism cells. Potassium, sodium, iron, magnesium, calcium, manganese, phosphorous, and other nutrients are included in the aqueous growth medium. These necessary materials may The carbon source, preferably the sole carbon source, for the fermentation process is an n-aliphatic hydrocarbon feed. The n-aliphatic hydrocarbon feed contains from potassium phosphate, potassium sulfate, potassium citrate,

Concentration (grams per liter) Usually Preferably Component Can use use use C11Cin n-aliphatic hydroearbom 4-120 5-80 10-50 Cellulose" 260 2. 5-40 5-25 K2HPO4 0. 5l5 l-lO 2-8 (NH4) 2111 04- 5-15 7-13 8-13 8804 0. 1-1. 0 0. 2-0. 9 0.3-0. 5 F0304 7 H20. 0 002-0. 5 0. 005-0. 04 0 0l-0. 02 MgSOr? H2O 0. l-0. 7 0. 2-0. 0. 3-0. 3 M11804- 7 H2O. 0. 002-0. 05 0. 005-0. 04 0. 01-0. 03 NaCl 0. 002-0. 05 0. 005-0. 04: 0. 01-0. 08 Water Remainder to equal wt. percent Cellulose is preferably in stri s such as about V to 2 Wide and to 6 long. Desirable strips are wide and 1 long.

Other optional mineral nutrients which may be included in trace amounts include:

Concentratioln (milligrams per iter) Usually Preferably Component Can use use use ZHSOJ'II2O 0-0. 4 0-0. 3 0-0. 2 0-0. 06 0-0. 05 0-0. 04 0-1. 2 0-0. 06 O-0. 2 0-0. 12 0-0. 005

The essential and optional nutrients may be supplied in the form of other salts or acids than those tabulated hereinabove.

A very satisfactory medium is prepared as follows:

To the above is added 10 cc./liter of a salt solution A prepared as follows:

Grams/liter Salt, solution A: distilled water MgSO -7H O 40 FeSO -7H O 2 MnSO -4H O 2 NaCl 2 The foregoing P medium has a pH of 7.8. A variation of the above is one in which phosphate is supplied in the form of phosphoric acid.

The temperature of the biosynthesis bath may be varied between about 20 C. and about 55 C. depending upon the specific microorganism being grown, but preferred temperatures when using bacteria are between about 25 C. and about 45 C. such as about 35 C. The pH is preferably in the range from 5.5 to 8.5 such as about 7.0. be supplied by any technique but are preferably supplied by their water-soluble salts.

Potassium may be supplied as potassium chloride, about 1 to 30 carbon atoms in the molecule preferably 11 to 30 carbon atoms. A desirable n-parafiin hydrocarbon feed contains from about 11 to 20 carbon atoms in the molecule, such as a feed of the following composition.

STRAIGHT CHAIN PARAFFIN MIXTURE Less than 0.01% aromatics.

The harvesting of the microbial cells and the amino acids accumulated in the fermentation broth can be done by suitable means. The cells may first be separated from the fermentation broth by centrifugation (e.g. closed bowl, liquid cyclones or hydroclones, evaporation (e.g. falling film, wiped film), filtration (e.g. micropore, dialysis, reverse osmosis), flocculation, settling and decanta tion (e.g. by adding flocculants, coagulants or filter aids or by changing pH or temperature) or any other method of separation or combination of methods, The separated, concentrated cells may then be dried by spray drying, drum drying, freeze drying, vacuum drying, tray drying, oven drying or any other drying procedure or combination of procedures to obtain a final product having extremely high protein content and no impurities detrimental to humans or animals.

The extracellular amino acids accumulated in the broth may be recovered by fractional crystallization or evaporation (e.g. falling film, wiped film), spray drying, drum drying, freeze drying, vacuum drying, tray drying, oven drying. Another general method of recovery would be the adsorption of the amino acids on ion exchange resins, followed by selective elution.

The cellulose is preferably cellulose acetate strips or cellulose bags which are prepared by treating wood pulp with acetic acid, acetic anhydride and sulfuric acid as a are secured by the addition of the cellulose strips or cellulose bags to the fermentation process in that the production of extracellular amino acids is markedly increased.

The present invention may be more readily understood by reference to the drawing which illustrates one embodiment of the same. Referring specifically to the drawing, a container or flask 10 contains a two hole stopper 1. Glass tubing 3 is inserted into the stopper 1, to which is attached at the upper end a dispo-plug 2 and at the lower end a cellulose bag 4. A concentrated P salt solution 5 is positioned within the cellulose bag 4 which is positioned within the saline solution plus substrate A6.

In order to further illustrate the invention, fermentations were carried out as illustrated in the following examples.

EXAMPLE 1 A sterile aerobic fermentation process was conducted in the apparatus illustrated in the drawing. 20 ml. of a sterile 5 concentrate of P salts medium were added into the cellulose bag. 80 ml. of a sterile 0.85% saline solution containing 2.0 ml. of substrate A was introduced into the flask. The saline solution was inoculated with a 1% inoculum of Micrococcus cerificans ATCC No. 14987, which had been grown on substrate A for 24 hours, and the fermentation was conducted on a rotary mechanical shaker at 300 rpm. at 30 C. for 72 hours. The fermentation broth was periodically sampled, at which time the bacterial cells and other impurities were removed by centrifugation, and the amount of extracellular amino acids in the clear centrifugate was determined by the following assay methods. One such series of assay methods is described in the second edition of Microbiological Assay of the Vitamin B-Complex and Amino Acids (1952) by E. C. Barton Wright, Pitman Publishing Corp., New York, NY. Another assay method used was the Technicon Auto Analyzer which provides an automatic analytical system based upon the ion exchange chromatography system established by Spackman, Moore and Stein.

The following results were obtained.

TABLE II Mg./l. amino acids Alanine 0 0 17 3, 729 78 2, 850 49 4, 610 Allo-isoleucine- 0 0 720 P 760 840 Arglnine O 0 18 1, 520 87 780 1, 810 Aspartic acid- 0 0 21 4, 120 109 2, 540 68 4, 610 Cysteic acid 0 0 260 210 260 Cystine 0 0 P P P Glutamic ac 0 0 24 7, 090 123 5, 640 87 8, 260 Glycine O 0 29 3, 070 144 2, 030 78 3, 160 Histid1ne 0 0 1, 300 P 970 P 2, 680 Isoleucine.-. 0 0 M. 1, 370 P 450 P 1, S30 Leuciue 0 0 2, 910 34 870 P 2, 390 Lysine 0 0 3, 650 P 2, 660 P 3, 970 Methionine. 0 0 80 P P P P Meth. sulfoxide 0 0 5 440 26 440 10 920 Ornithine 0 0 5, P 3, 640 2, Phenylalan 0 0 25 5 440 124 4, 680 268 3, 790 Pro1ine 0 0 240 950 1. 800 Serine 0 0 19 4, 260 94 1, 850 80 3, 250 Thrconine 0 0 7 2, 34 1, 240 P 2, 350 Tyrosine 0 0 590 P 100 920 Valine 0 0 10 2, 350 50 1, 230 P 2, 490

Total amino acids, mg./1 0 0 182 51, 370 902 33, 890 700 52,060

Fermentation Conditions:

Inoeulum M. cerificans (ml.) 0 1.0 1. 0 1.0 1.0 1.0 1. 0 1. 0 Substrate A (mL) 2. 0 0 2. 0 2. 0 2. O 2. 0 2. O 2. 0 Cellulose bag Yes Yes No Yes No Yes No Yes Fermentation time (days) 3 3 1 2 2 3 3 Noru.-P=Present trace amount.

EXAMPLE 2 catalyst. The cellulose is fully acetylated (three acetate groups per glucose unit) and, at the same time, the sulfuric acid causes degradation of the cellulose polymer so that the product contains only about 200 300 glucose units per polymer chain. At this point in the process the ce lulose acetate is partially hydrolyzed by the addition of water until an average of 22.5 acetate groups per glucose unit remain. This product is a thermoplastic.

Additional aerobic fermentation operations were conducted using cellulose strips. A series of three sterile fermentations was carried out in 500 ml. fermentation flasks containing 100 ml. of P medium, 2.0 ml. substrate A and 1.0 gram of cellulose strips A x 1"). The medium was inoculated with a 1% inoculum of Micrococcus cerificans ATCC :No. 14987. The fermentations were conducted As pointed out heretofore, unexpected, desirable results 75 on a rotary mechanical shaker at 300 rpm. at 30 C.

The fermentation flasks were periodically removed at 1, 2 and 3 days and the broth harvested and assayed as in Example 1. The results obtained are set forth in Table III.

TABLE III One-day Two-day Three-day fermentation fermentation fermentation Amino acid mgJl. mg./l. Inga/l.

Allo-isoleucine 868 758 584 Ala 2, 555 3, 610 2, 710 854 869 380 2, 932 3, 758 1, 860 202 905 562 1, 619 4, 635 3, 116 1, 014 4, 733 2, 783 1, 056 1, 286 374 1,321 1, 152 977 662 907 246 2, 398 1, 444 1, 577 4, 071 5, 735 3, 235 731 924 360 1, 332 1, 690 1, 090 243 523 124 1, 345 1, 243 553 From the foregoing, it is evident that cellulose (strips or bags) substantially increase the yields of high quality amino acids.

What is claimed is:

1. Aerobic fermentation process for the production of extracellular amino acids which comprises incubating a fermentation broth with Micrococcus cerificans ATCC No. 14987, said broth comprising an aqueous inorganic salt growth medium, an oxygen-containing gas and a liquid petroleum hydrocarbon fraction as a primary source of carbon, conducting the fermentation process in the presence of cellulose acetate and under conditions adapted to promote cell growth whereby a high yield of extracellular amino acids is secured.

2. Process as defined by claim 1 wherein said hydrocarbon fraction comprises essentially C -C normal parafiins.

3. Process as defined by claim 2 wherein said hydrocarbon fraction comprises C C normal paraffins and wherein the C-C17 fraction is in excess of 80% by weight.

4. Process as defined by claim 1 wherein the cellulose is selected from the class consisting of cellulose acetate strips and cellulose acetate bags.

5. Process as defined by claim 1 wherein the pH is maintained in the range from 5.0 to 8.5 and wherein the temperature is maintained in the range from about C. to C.

References Cited UNITED STATES PATENTS 3,406,095 10/1968 Otsuka et al -28 LIONEL M. SHAPIRO, Primary Examiner M. D. HENSLEY, Assistant Examiner US. Cl. X.R. 

